Acid is a potential interferent in fluorescent sensing of chemical warfare agent vapors

The study addresses whether fluorescence changes observed in solid-state films designed to detect G-series organophosphorus nerve agents (e.g., Sarin) arise from the intended nucleophilic substitution at phosphorus or from acid-induced protonation of basic sensing motifs. Many reported chemosensors employ pyridyl or related nitrogen-containing nucleophiles to trigger fluorescence upon reaction with agents or simulants such as diethylchlorophosphate (DCP) and diisopropyl fluorophosphate (DFP). However, G-series agents can contain or generate acids (e.g., hydrofluoric acid from hydrolysis), and nitrogen nucleophiles are basic, raising the possibility of acid interferents causing false positives. The purpose is to disentangle acid-driven protonation effects from true phosphorylation/cyclisation mechanisms in film-based fluorescence sensing, using pyridyl-containing materials and a thiazolyl analogue to probe acid susceptibility and response under controlled analyte purity and storage histories. This has significance for developing selective, field-deployable sensors and for interpreting prior literature that may not have controlled for acid impurities.

Fluorescence-based sensing has been widely explored for nerve agent detection, inspired by successes with explosives. Organic nucleophiles (notably pyridyl, quinoline, quinoxaline) and organometallic systems have been designed to react with electrophilic phosphorus centers to induce spectral changes. Many reports on G-series detection use DCP and DFP as simulants and assume formation of pyridinium salts via phosphorylation followed by hydrolysis, often without kinetic or intermediate evidence, and frequently without specifying simulant purity. DFP and DCP are known to hydrolyze, generating acids (HF for fluorophosphates; HCl for chlorophosphates). Literature indicates DFP hydrolyzes over hours (accelerated by glass), while DCP hydrolyzes faster due to a weaker P–Cl bond. Prior 31P NMR studies suggest pyridine reacts slowly with diethyl chlorophosphate with equilibrium to the left and faster changes in the presence of water, questioning rapid solid-state reactions. Overall, selectivity and false positives, especially from acids, remain under-addressed.

• Simulant preparation and stability: DFP synthesized from diisopropylphosphite, cupric chloride, and cesium fluoride, purified by Kügelrohr distillation. Storage conditions: sealed plastic at −20 °C for 14 days (non-treated), 143 days (aged), or over hexamethylenetetramine (HMTA, acid scavenger; 20 mg per 200 µL) for 240 days at −20 °C. DCP freshly distilled, stored in nitrogen-filled glovebox; stability in capped glass vials under ambient lab conditions (RH ~50–60%) was monitored.
• NMR characterization: 31P NMR in CD2Cl2 to assess simulant hydrolysis and acid formation. For DFP, fresh samples showed δ = −10.71 ppm (doublet, JPF = 972.8 Hz); aged samples exhibited an additional δ = −0.31 ppm peak (diisopropyl phosphoric acid). DCP hydrolysis tracked over 1 h, 8 h, 3 d showing phosphate, pyrophosphate, and polyphosphate signals. Acid removal tested with anhydrous K2CO3 (10 wt%) pretreatment.
• Film preparation: Sensing compounds (including pyridyl and thiazolyl systems 1, 2, and pyridyl derivatives 3 and 4) blended with cellulose acetate (CA). Solutions: sensing compound in ethanol (1–10 mg/mL); CA in acetone (20 mg/mL). Mix 200 µL sensing solution with 900 µL CA solution; spin coat on fused silica at 2000 rpm for 60 s (thickness ≈220 nm).
• Optical measurements: Films excited at 365 nm (LED). Photoluminescence (PL) spectra recorded with OceanOptics Flame or FS5 spectrometers; absorption and excitation spectra measured to compare with reference compounds.
• Vapor exposure protocols: In air experiments, 2 µL of DFP, DCP, HCl(aq, 16%), or a droplet of Sarin placed in chamber bottom to generate vapor at 20–22 °C. For DCP under inert conditions, nitrogen bubbled through DCP (with/without anhydrous K2CO3 pretreatment) and delivered to chamber; effluent scrubbed in 20 wt% NaOH.
• Chemical controls and reference compounds: Solution 1H NMR (CD2Cl2) of compounds 1 and 2 with 1 equiv. freshly prepared, acid-free DFP over 0.5–25 h to probe for phosphorylation/cyclisation. Synthesis of protonated and cyclised references: 1-H+ (X = Cl), cyclised 1′ (X = Br), 3 and 3-H+ (X = Cl), and cyclised 2′ (X = Br). Films of these references in CA prepared at appropriate wt% (typically 2–10 wt%, constrained by solubility/optical clarity). Comparisons of absorption, PL, and excitation spectra of films exposed to aged/treated simulants or HCl versus reference films.
• Simulant treatments to remove acid: DFP treated with HMTA or poly(4-vinylpyridine) (PVP); DCP treated with anhydrous K2CO3. Response kinetics compared for non-treated versus treated simulants.
• Sarin tests: Films of 1 and 4 exposed to fresh Sarin (prepared and twice-distilled less than one week prior) versus aged Sarin (stored >6 months), under the same chamber conditions.

• Acid impurities in G-series analytes drive rapid fluorescence responses in pyridyl-based sensing films; acid-free analytes do not: • DFP: Fresh/acid-free DFP (HMTA- or K2CO3-treated) caused no observable change in absorption or PL of films containing sensing compounds (e.g., 1:CA). Aged DFP (143 days at −20 °C) induced strong PL turn-on; non-treated DFP (14 days) gave intermediate response. 31P NMR confirmed hydrolysis/acid presence: fresh DFP δ = −10.71 ppm (JPF = 972.8 Hz); aged DFP showed additional δ = −0.31 ppm (diisopropyl phosphoric acid). HMTA-treated DFP showed no change even after 8 months. • DCP: Even when glovebox-stored, non-treated DCP vapor produced rapid PL turn-on in 1:CA films within ~30 s; K2CO3-treated DCP gave no PL response under nitrogen. In air, freshly distilled DCP produced a slow PL increase; pre-exposure of DCP to air for 30–60 min led to rapid PL response, consistent with fast hydrolysis and HCl formation.
• Mechanism is protonation, not phosphorylation/cyclisation: • Solution 1H NMR of 1 and 2 with 1 equiv. acid-free DFP showed no new peaks attributable to cyclised products after 25 h, contradicting the assumption of rapid phosphorylation/cyclisation. • Film spectra (absorption, PL, excitation) of 1:CA exposed to aged DFP or non-treated DCP matched those of protonated references (1-H+, 3-H+) and differed from cyclised 1′. For 2, excitation spectra of aged DFP/HCl-exposed films matched protonated 2, not cyclised 2′.
• Acid scavenging removes responses: Treating DFP with HMTA or PVP, and DCP with K2CO3, abolished (or greatly slowed) fluorescence responses in films of compounds 1, 3, and 4.
• Sarin behavior mirrors simulants: Aged Sarin induced PL turn-on in films of 1 and 4; freshly synthesized/distilled Sarin caused no response, consistent with acid impurities (e.g., HF) driving the signal.
• Sensitivity to acid: In solution, sensing materials detect acid down to 0.4 µM (reported in Supplementary Information).
• Additional quantitative/parametric details: pKa of conjugate acid of thiazole ~2.5 versus pyridine ~5.2 (thiazolyl unit less susceptible to acid). Film thickness ≈220 nm. Storage histories: DFP tested fresh, 14 days, 143 days; HMTA treatment for 240 days without degradation.

The findings directly address the central question of whether fluorescence-based solid-state films detect G-series nerve agents via intended nucleophilic phosphorylation or via acid interferents. Across multiple pyridyl-containing systems (with or without alcohol functions) and a thiazolyl analogue, rapid fluorescence turn-on correlated with the presence of acid impurities in Sarin, DFP, and DCP. Acid removal through scavengers (HMTA, PVP) or base treatment (K2CO3) eliminated or greatly slowed responses. Spectroscopic fingerprints in films matched protonated species rather than cyclised products, and solution NMR showed negligible phosphorylation/cyclisation over 25 h with acid-free DFP. These results demonstrate that in practical, solid-state sensing conditions, fast responses are dominated by protonation due to acid contamination/hydrolysis products rather than the slower multistep processes of diffusion, phosphorylation, and hydrolysis. This has major implications for the selectivity and reliability of many reported sensors that rely on basic nucleophilic motifs, underscoring the need for rigorous analyte purity control and for sensor designs that are insensitive to acids or that can discriminate between acid and nerve agents. The work also suggests that previously reported rapid film responses to G-series simulants may require re-interpretation as acid detection, particularly when experiments were performed in ambient air where DCP/DFP hydrolyze readily.

This study shows that the fast fluorescence responses of pyridyl-based (and related) sensing films to Sarin, DFP, and DCP vapors arise from protonation by acid impurities rather than phosphorylation/cyclisation mechanisms. Acid-free analytes do not produce prompt film responses, and spectral analyses match protonated reference compounds. The work highlights the prevalence and impact of acid impurities from analyte synthesis, storage, and rapid hydrolysis (especially for DCP in air), and establishes that acid false positives likely underlie many reported solid-state sensing results for G-series agents and simulants. Future work should focus on: (i) developing sensing chemistries that are selective for organophosphorus electrophiles while resisting acid-induced responses; (ii) implementing standardized simulant purification, storage, and verification (e.g., in situ 31P NMR, acid scavengers) in sensor testing protocols; (iii) designing ratiometric or multi-analyte sensing schemes that differentiate acid from phosphorylation events; and (iv) validating sensors with rigorously fresh, acid-free agents under controlled atmospheres and realistic field conditions.

The study centers on a subset of sensing motifs (pyridyl, thiazolyl derivatives) in cellulose acetate films; results may not generalize to all sensor architectures or matrices. Acid levels in analytes were inferred via 31P NMR hydrolysis products and response differences but not quantified systematically for every test. Kinetic analyses emphasize rapid, qualitative PL changes rather than detailed rate constants. Experiments focus on DFP and DCP simulants and Sarin under laboratory conditions; broader agent classes and environmental variables (temperature, humidity, sorbent substrates) were not exhaustively explored.